JP6452115B2 - Cholesterol storage disease treatment - Google Patents

Description

  The present invention relates to pharmaceutical compositions and methods for treating diseases caused by cholesterol accumulation, such as lysosomal disease. More specifically, the present invention relates to a pharmaceutical composition for treating a disease caused by cholesterol accumulation, comprising hydroxypropyl-γ-cyclodextrin as an active ingredient. The present invention also relates to a method for screening for therapeutic agents for diseases caused by cholesterol accumulation, such as lysosomal disease.

  If an enzyme related to lysosome, which is one of the organelles in the cell, is genetically deleted or mutated, the substance to be decomposed or transported accumulates as a foreign substance inside and outside the cell. An inborn error of metabolism caused by such a phenomenon is known as lysosome disease. Examples of lysosomal diseases include Niemann-Pick disease and GM1 gangliosidosis.

  Niemann-Pick disease type C (NPC) is one of congenital lysosomal diseases caused by abnormalities in the membrane protein NPC1 that controls the transport of lipids, mainly cholesterol, in the cells, or in the secretory protein NPC2 molecule that coexists with NPC1 in the endosome. It is. In patient cells, free cholesterol and lipid accumulate in lysosomes. Characterized by liver, spleen enlargement and neurological symptoms. It is a rare intractable disease that develops in early childhood, causes hepatosplenomegaly and progressive neuropathy, and dies around 10 years old. An effective treatment for this disease has not been established.

Cyclic oligosaccharide cyclodextrins (CyDs) are unimolecular host molecules having a hydrophobic cavity in the molecule. When a guest molecule is incorporated into a cavity of CyDs to form an inclusion complex, the physicochemical properties of the guest molecule change variously. The supramolecular inclusion phenomenon of CyDs called molecular capsules is effectively used in many fields. Especially in drug development,
It is widely applied to improve formulation characteristics and to build drug delivery systems.
Recently, Liu et al. Showed that when 2-hydroxypropyl-β-cyclodextrin (HPBCD) was intravenously administered to Npc1 gene-deficient (Npc1 − / −) mice, it was effective in improving the disease state and prolonging life. It has been reported that when administered directly, the improvement effect increases several hundred times as compared with systemic administration (Non-patent Document 1). Based on the results of these basic studies, the US FDA has approved humane use of HPBCD (intravenous and intrathecal) for children with NPC. Against this backdrop, in Japan, HPBCD injections were prepared in-house at Saga University Hospital and treatment for children with NPC began. As a result of continuing intravenous infusion of HPBCD (1500 mg / kg once to 1-3 times a week) over 1 year to children with NPC, certain effects were obtained in reducing hepatosplenomegaly and improving brain waves in children However, it has not improved neurological symptoms. Therefore, in addition to HPBCD, a glycolipid synthesis inhibitor miglustat (50 or 100 mg at a time, twice a day) was used in combination. Furthermore, in order to deliver HPBCD directly into the brain without going through the blood-brain barrier, intrathecal administration and intraventricular administration via an Ommaya reservoir (30 mg / kg, weekly 1) concurrently with intravenous administration of HPBCD Times). Treatment with HPBCD is the first in Japan, and there is no precedent for large-dose or long-term administration, so treatment is continued while examining the effectiveness of treatment and adverse events. However, due to side effects, it has not been generalized in Japan.

  On the other hand, HPBCD has been recognized as a pharmaceutical additive (dissolution aid), but there is a concern about kidney damage. In addition, events such as pulmonary disorders have been reported, and in the case of large doses or long-term administration, its safety is a problem. Therefore, there is a need for a safer therapeutic agent for NPC that replaces HPBCD.

  GM1 gangliosidosis is one of Gaucher's disease whose pathogenesis is caused by a mutation of lysosomal-β-glucosidase, which is a sugar hydrolase, and a mutation of lysosomal-β-galactosidase. A disease in which beta-galactosidase deficiency causes glycolipids such as GM1-ganglioside and asialo GM1-ganglioside, which are the substrates, to accumulate in the brain and internal organs (liver, spleen), and mucopolysaccharides such as keratan sulfate in the bone. is there. The disease develops in early infancy, including spastic paraplegia, widespread central nervous system disorders, cherry red spot on the fundus, hepatosplenomegaly, infant type with bone abnormalities (type 1), and onset from early childhood, with progressive central neuropathy There are three types: a young type (type 2), and an adult type (type 3) in which symptoms such as dysarthria appear from the schoolchildhood and extrapyramidal symptoms are the main.

  Enzyme replacement therapy has been the main treatment to date for these diseases, but enzyme preparations are difficult to reach the central nervous system and have no therapeutic effect on the nervous system including the brain. The problem is that the infusion treatment of the preparation must be continued for a lifetime. Therefore, new therapeutic agents for these lysosomal diseases are desired.

  Artificial pluripotent stem cells (iPS cells) that are artificially produced from human somatic cells can proliferate indefinitely and have pluripotency (ie, the ability to generate various cell types in vitro). It can be guided as shown. These features make iPS cells have potential uses as a resource for cell therapy in clinical medicine. The process of iPS cell generation known as reprogramming is triggered by the expression of four transcription factors, OCT3 / 4, Sox2, Klf4, and c-Myc. These factors are the same as the core factors underlying the pluripotency of other pluripotent stem cells such as embryonic stem cells (ES cells). Overexpression of these four factors in human skin-derived fibroblasts was mediated early by lentivirus and retroviral vectors. Although these gene expression systems are stable, there were two potential problems. One is that the genes encoding the four factors are integrated into the host genome, and the other is that they remain in the iPS cells from which they were obtained. Therefore, there is a risk of insertion mutation that can promote tumor formation in vivo.

  Therefore, an efficient and safe reprogramming method based on Cre / loxP based on a recombination system, adenovirus vector, piggyback transposon, microRNA, or protein has been developed. There were problems with the necessity of repeated induction, and the short length of foreign genes retained in the host genome. Recent studies have reported the production of iPS cells from blood cells using episomal plasmid vectors that rarely integrate into the host genome, but with low efficiency (˜0.1%), In addition to the four reprogramming factors, p53 knockdown and transient expression of EBNA are required.

Sendai virus (SeV) vector technology is an alternative strategy developed to overcome the above problems. Sendai virus vector is a minus-strand RNA that expresses the gene of interest without being integrated into the host genome, and has been used to efficiently produce iPS cells from human skin-derived fibroblasts and blood cells. (Non-patent document 2, Non-patent document 3). The frequency of iPS cell colonies generated by Sendai virus vectors is higher than that achieved by conventional methods using retrovirus or lentiviral vectors (0.1% vs. 0.01%). However, since Sendai virus remains in cells for over a month, it takes a long time to produce iPS cells that do not contain a transgene. In recent years, a temperature-sensitive Sendai virus (TS-SeV) system has been developed to prevent the production of uncontrollable iPS cells due to the continuous cell replication of Sendai virus (Non-Patent Document 4). Ts-SeV can be easily and immediately removed from umbilical cord blood cells or fibroblast-derived iPS cells by increasing the temperature, but the production efficiency of iPS cells is lower than that of SeV.
In addition, a method for producing iPS cells from peripheral blood monocytes using SeV has been reported. There, SeV vectors that continuously express the reprogramming genes Oct4, Sox2, Klf4, and c-Myc are used, and the siRNA is used to remove the reprogramming gene-containing viral vector from the cells. (Patent Document 1).

  Dermal fibroblasts are the most common cell type used to generate iPS cells, but skin biopsies are invasive and not ideal for children or patients with skin diseases or coagulopathy. Peripheral blood cells are the preferred source cells, but iPS cell production from peripheral blood cells using Ts-SeV vectors has not been reported, and long-term retention of Sendai virus in iPS cells The problem remains when using sensitive SeV.

  A number of iPS cell lines derived from somatic cells of patients carrying pathogenic mutations, obtained using methods involving Sendai virus, have been shown to replicate the disease phenotype. Thus, these cell lines are powerful tools not only for cell therapy but also for biomedical research and drug development. Biomaterial samples from patients with intractable diseases are essential to study the molecular mechanisms of disease and develop new therapeutics, but the number of samples from such patients is usually limited, Disease-derived iPS cells are expected to be useful as an alternative or supplemental source of biomaterials for cell therapy. Thus, iPS cells are currently used as cell sources and disease cell models, but their use is limited by inefficient production and the presence of transgenes in the cells. Therefore, a safe iPS cell production method that can be produced more efficiently and does not contain a transgene in the cell is desired.

WO2012 / 063817

Liu et al., Proc Natl Acad Sci USA, 106, 2377 (2009) Fukiki et al., Proc. Jpn. Acad. Ser. B, Phys. Biol. Sci. 85, 348-362, 2009 Seki et al., Cell Stem Cell 7, 11-14, 2010 Ban et al., Proc. Natl. Acad. Sci. USA 108, 14234-14239, 2011 Irie et al., J. Phar. Sci., Vol 86, No. 2, pp. 147-162, 1997

An object of the present invention is to provide a pharmaceutical composition for treating diseases caused by cholesterol accumulation such as lysosomal disease, such as Niemann-Pick disease and GM1 gangliosidosis.
The present invention also aims to provide a method for screening those pharmaceutical compositions, more specifically, an iPS cell line that replicates a phenotype of a disease caused by cholesterol accumulation such as lysosomal disease. It is an object of the present invention to provide a method for screening a used pharmaceutical composition for treating the disease.
The present invention also relates to a method for efficiently producing an iPS cell line used in the screening method, and more specifically, an iPS used in the screening method using a temperature-sensitive Sendai virus having only a specific reprogramming factor. The present invention relates to a method for efficiently producing a cell line.
It is also an object of the present invention to provide an iPS cell line that does not contain a transgene, which is an effective cell model for intractable diseases.

The present inventors have developed a new Sendai virus vector, TS12KOS, which has improved iPS cell production efficiency and can be easily removed from cells without being incorporated into intracellular DNA. The present inventors also use the TS12KOS vector to produce an iPS cell line showing a phenotype of the disease from a patient with an intractable disease, and to screen for a therapeutic drug candidate for the disease using the cell line. Developed. The present inventors have further found a pharmaceutical composition for treating diseases caused by cholesterol accumulation such as lysosomal disease such as Niemann-Pick disease and GM1 gangliosidosis using such a method.
The present invention includes the following.

(1) A pharmaceutical composition for treating or preventing lysosomal disease, comprising hydroxypropyl-γ-cyclodextrin as an active ingredient.
(2) The pharmaceutical composition according to (1), wherein the lysosomal disease is Niemann-Pick disease.
(3) The pharmaceutical composition according to (1), wherein the lysosomal disease is GM1 gangliosidosis.
(4) The pharmaceutical composition according to any one of (1) to (3), wherein the pharmaceutical composition is an injection and is administered for a long time.

(5) A method for screening drug candidates for intractable diseases,
The following steps: (i) a step of infecting a cell derived from a patient with an intractable disease with a temperature sensitive Sendai virus vector to initialize the cell, wherein the vector comprises an NP gene, an alanine residue (D433A, R434A, and K437A), each of which includes a P gene, a M gene, an HN gene, and an L gene containing three mutations. Between the P gene and the M gene, three reprogramming genes, KLF4, OCT3 / 4, And (ii) culturing the cells infected with the vector at a temperature exceeding 37 ° C., thereby removing the vector carrying the reprogramming gene from the cells. An iPS cell produced by the step of producing an iPS cell comprising a step of producing an iPS cell containing no transgene After differentiating into lineage, cultured with the target substance, and then, detecting the influence of the target substance on the cells,
A screening method comprising:
(6) The screening method according to (5), wherein the culture in the step (ii) is 38 ° C. ± 0.5 ° C.
(7) The screening method according to (5) or (6), wherein the cells derived from a patient with an intractable disease are skin fibroblasts.
(8) The screening method according to (5) or (6), wherein the cells derived from a patient with an intractable disease are cells derived from peripheral blood.
(9) The screening method according to any one of (5) to (8), wherein the intractable disease is lysosomal disease.
(10) The screening method according to (9), wherein the lysosomal disease is Niemann-Pick disease or GM1 gangliosidosis.

(11) (i) A step of infecting a cell derived from a lysosomal disease patient with a temperature-sensitive Sendai virus vector to initialize the cell, wherein the vector comprises an NP gene, an alanine residue (D433A, R434A, And K437A), each of the P gene, the M gene, the HN gene, and the L gene including three mutations. Between the P gene and the M gene, three reprogramming genes, KLF4, OCT3 / 4, and SOX2 And (ii) culturing cells infected with the vector at a temperature exceeding 37 ° C. to remove the vector carrying the reprogramming gene from the cells. A step of producing an iPS cell containing no transgene,
An iPS cell derived from a patient with lysosomal disease produced by a process comprising:
(12) The iPS cell according to (11), wherein the cell derived from a patient with lysosomal disease is a skin fibroblast.
(13) The iPS cell according to (11), wherein the cell derived from a patient with lysosome disease is a cell derived from peripheral blood.
(14) The iPS cell according to any one of (11) to (13), wherein the lysosomal disease is Niemann-Pick disease or GM1 gangliosidosis.
(15) The iPS cell according to (14), wherein the lysosomal disease is Niemann-Pick disease and has mutations in the NPC1 gene and the NPC2 gene.
(16) When the lysosomal disease is Niemann-Pick disease and differentiated into hepatocyte-like cells, the following phenotype:
(A) increased intracellular cholesterol accumulation,
(B) the autophagy function of the cell is impaired, and (c) ATP production in the cell is reduced,
The iPS cell according to (15), wherein

The composition containing the hydroxypropyl-γ-cyclodextrin of the present invention as an active ingredient is effective against lysosomal diseases, particularly iPS cells that replicate the phenotype of Niemann-Pick disease or GM-1 gangliosidosis, Moreover, it is effective as a therapeutic agent for those diseases.
In addition, iPS cells can be efficiently produced from cells from patients with intractable diseases by the method using the temperature-sensitive Sendai virus vector of the present invention. The produced iPS cells replicate the disease phenotype and do not contain a transgene. . By using this iPS cell, drug candidates for diseases can be easily screened. Furthermore, the produced iPS cells themselves are safe without becoming cancerous.

A comparison of the schematic structure of a conventional vector and the temperature-sensitive Sendai virus (TS-SeV) vector of the present invention, TS12KOS, is shown. The TS12KOS vector contains three mutations in the RNA polymerase-related gene (P) and has coding sequences for KLF4 (K), OCT3 / 4 (O), and SOX2 (S) in the KOS direction. The HNL / TS15 c-Myc vector has two additional mutations in the large polymerase (L) gene, L1361C and L1558I, and an exogenous c- inserted between the hemagglutinin-neuraminidase (HN) gene and the L gene. It has a Myc cDNA sequence. Conventional vectors have three reprogramming factors in each vector. The result of producing iPS cells from human skin-derived fibroblasts is shown. N1, N2, and N3 represent individual healthy volunteers. Experiments were performed in triplicate (mean ± SD). * P <0.01, TS12KOS vector vs. conventional vector, Student's t test. The period during which the temperature change from 37 ° C. to 36 ° C. in iPS cell production is shown. Data are the mean ± SD of each of 3 experiments. _{** P <0.02, # P <} 0.05, t -test of Student. The result of the nested RT-PCR analysis of SeV vector removal after the temperature change from 37 degreeC to 38 degreeC in a human fibroblast-derived iPS cell is shown. The number of clones from which the vector after passage (1 or 2) was removed is shown. The result of iPS cell preparation from human peripheral blood cells is shown. Experiments were performed in triplicate (mean ± SD). (A) N1, N2, and N3 represent individual healthy volunteers. * P <0.01, TS12KOS vector vs. conventional vector, Student's t test. (B) The result of the nested RT-PCR analysis of SeV vector removal after the temperature change from 37 degreeC to 38 degreeC is shown. The number of clones from which the vector after passage (1 or 2) was removed is shown. The tissue morphology of a representative teratoma derived from an iPS cell line formed with the TS12KOS vector is shown. It is the result of hematoxylin and eosin staining. All three germ layer progeny were observed in teratomas. The iPS cell line is derived from human fibroblasts, BJ. CE: cubic epithelium (ectoderm), G: glandular structure (endoderm), M: muscle tissue (mesoderm), C: cartilage (mesoderm). The scale bar is 100 μm. The phase contrast image of the iPS cell line derived from a Niemann-Pick disease type C patient is shown. Immunofluorescence and alkaline phosphatase (AP) staining. The iPS cell lines, NPC5-1 and NPC5-2, and NPC6-1 and NPC6-2 are derived from NPC patients and NPC6, respectively. The scale bar is 200 μm. The results of RT-PCR analysis of Sendai virus and human ES cell markers of iPS cells from Niemann-Pick disease type C patients are shown. NPC5 and NPC6 are derived from NPC5 and NPC6, which are NPC patients, respectively. 201B7: Control human iPS cell line. SeV (+): SeV-infected human fibroblasts on day 7. SeV: First RT-PCR of Sendai virus. Nested: Nested RT-PCR for Sendai virus. Histological analysis of teratomas derived from NPC-iPSC. The results of hematoxylin and eosin staining are shown. CE: cubic epithelium (ectoderm), G: glandular structure (endoderm), M: muscle tissue (mesoderm), C: cartilage (mesoderm), MP: melanin pigment (ectoderm). The scale bar is 100 μm. Confirmation of mutation in NPC1 gene of NPC-derived iPS cell line. Mutations 2000 C> T (S667L) and 3482 G> A (C1161L) were observed in the iPS cell line from patient NPC5 (left panel). On the other hand, in the iPS cell line from patient NPC6, both 3263 A> G (Y1088C) and a short deletion mutation in which the 581 to 592 nucleotide region was replaced with a G residue causing a frameshift (right) panel). Mutations are indicated by arrows. It shows the path of differentiation of NPC-derived iPS cells into hepatocyte-like cells. The pathway is divided into three periods: endoderm differentiation from day 0 to day 4, liver differentiation from day 4 to day 11, and liver maturation from day 11 to day 18. The culture conditions are described below for each period. Cells were harvested on both days 4 and 11 and replated under the conditions described. The cell size of HLCs derived from NPC-iPS cells is shown. Figure 5 shows cholesterol accumulation in HLCs derived from NPC-iPS cells. Free cholesterol was detected by Filipino staining (upper panel) and relative intensity was calculated for the normal iPS cell line, N1-12 (lower graph). Data are the mean ± SD of 3 independent experiments. * P <0.01, NPC-iPS cell line in the figure vs. iPS cell line in the figure, Student's t-test. The scale bar is 100 μm. ATP levels in iLC cell line derived HLCs are shown. Experiments were performed in triplicate (mean ± SD). * P <0.01, _# P is <0.05, normal iPS cell lines in NPC-iPS cell lines versus figures in the drawing. Student's t test. The expression of microtubule associated protein 1 light chain 3 (LC3) is shown. _{* P <0.01, # P <} 0.05, NPC-iPS cell lines versus normal iPS cell lines in Fig, N1-12 and N3-2. Student's t test. Expression levels were normalized to α-tubulin expression in each iPS cell line. The expression level of insoluble p62 is shown. It is a result of immunofluorescence staining of P62. Abnormal aggregation of P62 was significantly present in NPC-derived HLCs (upper panel). Aggregated granules were counted and the results summarized in the figure (lower panel). The proportion of cells with more than 40 granules was increased with NPC-derived HLCs compared to normal HLCs. Nuclear staining: Hoechst 33258. Scale bar is 25 μm. The result which confirmed the effect of a series of hydroxypropyl cyclodextrin with respect to the reduction | restoration of free cholesterol accumulation in NPC origin HLCs is shown. The results of Philippine staining are shown in the upper panel, and the analysis results of IN CELL ANALYZER are shown in the lower panel. Data are the mean ± SD of 3 independent experiments. _{* P <0.01, # P <} 0.05. Untreated vs. treatment of each NPC-derived HLC. Student's t test. Scale bar is 50 μm. FIG. 5 shows the dose effect of HPBCD and HPGCD on reducing free cholesterol accumulation in NPC-derived HLCs. * P <0.01. Untreated vs. treatment of each NPC-derived HLC. Student's t test. The result of having confirmed the effect of HPBCD with respect to reduction of free cholesterol accumulation in the differentiation of NPC origin iPS cell is shown. The experimental design is shown in the upper panel, and the results of Philippine staining are shown in the lower graph. Experiments were performed in triplicate (mean ± SD). _{* P <0.01, # P <} 0.05. Non-treatment versus treatment. Student's t test. It is the result of having confirmed the effect of the hydroxypropyl cyclodextrin with respect to the ATP level of HLCs derived from a NPC-iPS cell line. Data are the mean ± SD of 3 independent experiments. _# P <0.05. Non-treatment versus treatment. Student's t test. It is the result which confirmed the effect of the hydroxypropyl cyclodextrin with respect to the LC3 expression level of HLCs derived from a NPC-iPS cell line. Treated with 1 mM HPBCD for 4 days. Treated with 1 mM HPGCD for 4 days. Expression levels were normalized by α-tubulin expression in each iPS cell line. It is the result which confirmed the effect of the hydroxypropyl cyclodextrin with respect to the p62 expression level of HLCs derived from a NPC-iPS cell line. It is the figure which showed the ratio of HLCs with an insoluble P62 aggregate at the time of treating HLCs derived from NPC-iPS cell line with hydroxypropylcyclodextrin. β: treated with 1 mM HPBCD for 4 days. γ: treated with 1 mM HPGCD for 4 days. It is the result of carrying out cluster analysis of HLCs derived from the NPC-iPS cell line treated with hydroxypropylcyclodextrin. It is a result of principal component analysis (PCA) of HLCs derived from NPC-iPS cell line treated with hydroxypropylcyclodextrin. The result of the microarray analysis which compared normal person origin HLC and NPC origin HLC is shown. Molecules surrounded by a square are signature molecules that are significantly altered in NPC-derived HLC compared to normal human-derived HLC (p <0.05). The upper panel shows molecules that are significantly down-regulated in NPC, and the lower panel shows molecules that are significantly up-regulated in NPC. The result of the microarray analysis which considered the influence by HPCBD or HPGCD processing is shown. The upper panel shows the effect of HPBCD processing, and the lower panel shows the effect of HPGCD processing. Molecules surrounded by a square are signature molecules that are significantly changed by HPCBD or HPGCD treatment (p <0.05). The result of the hierarchical cluster analysis of the gene which changed notably by HPCBD or HPGCD processing is shown. The left panel shows a gene containing a signature molecule changed by HPBCD treatment, and the right panel shows a gene containing a signature molecule changed by HPGCD treatment. It is the result of having confirmed the serum marker (ALT and AST) of the model mouse (NPC mouse) which processed HPGCD. It is a histological analysis result of the liver of a model mouse (NPC mouse) treated with HPGCD. The arrow indicates a state where fat is accumulated. Upper panel (x200), lower panel (x400). Scale bar: 50 μm. It is a histological analysis result of the cerebellum of a model mouse (NPC mouse) treated with HPGCD. Depletion of Prikinje cells (arrow) has been recovered by HPGCD treatment. The upper panel is H & E stained, and the lower panel is immunostained with calbindin. It is the result of confirming the effect of HPGCD treatment on the survival of model mice (NPC mice). It is the result of having tested the acute toxicity of HPBCG and HPGCD.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the embodiments described below.
The vector carrying the reprogramming gene used for producing iPS cells in the present invention has a NP gene, a P gene and an L gene derived from Sendai virus (SeV), and a temperature sensitive Sendai virus in which the F gene is deleted. (TS-SeV) vector. Included within the L gene are three mutations that result in alanine residues (D433A, R434A, and K437A). The Sendai virus vector (SeV) having these three mutations is characterized by moderate expression of GFP at 37 ° C, but weak expression at temperatures above 38 ° C. The vector used in the present invention further includes a sequence encoding three reprogramming genes, KLF4 (K), OCT3 / 4 (O), and SOX2 (S) between the P gene and the M gene in the KOS direction. And mounting. As a result, even cells derived from patients with refractory diseases can be efficiently initialized and viral vectors can be easily removed.

  The Sendai virus vector is a gene introduction / expression vector that can express an arbitrary gene by inserting the gene into the Sendai virus genome or replacing the Sendai virus gene with an arbitrary gene. Sendai virus has NP, P, M, HN, F and L genes, and the NP, P and L genes of the virus are genes involved in Sendai virus transcription and replication, while M, F and The NH gene is a gene involved in virus particle formation. Therefore, Sendai virus vector deficient in F gene cannot form new virus particles alone after infecting cells, and becomes non-transmissible.

  The TS-Sev vector used in the present invention has three mutations in the L gene that contribute to temperature sensitivity, and three reprogramming genes loaded between the P gene and the M gene in the KOS direction, KLF4 (K ), OCT3 / 4 (O), and SOX2 (S), as long as it has the property of lacking the ability to form virus particles, Sendai virus with other mutations and changes can be used. is there.

The reprogramming gene to be inserted into SeV used in the present invention is characterized in that it contains Oct3 / 4, Sox2, and Klf4 genes of human, mouse or any mammal in the order of specific sequences. In addition, other reprogramming genes involved in tumor formation, for example, c-Myc gene and L-Myc gene may be included, but preferably, the reprogramming genes include only Oct3 / 4, Sox2, and Klf4, It does not contain any other genes with tumorigenic activity.
The TS-SeV vector having the above characteristics can be prepared using a known method.

For cell reprogramming, a TS-SeV vector carrying a reprogramming gene in the form of a virus particle is infected with a differentiated cell to be reprogrammed. The cells to be initialized are cells derived from patients with intractable diseases, preferably skin fibroblasts or cells derived from peripheral blood. Peripheral blood-derived cells may be either T lymphocyte cells or monocyte cells. Peripheral blood is less invasive than dermal fibroblasts and is also suitable for pediatric patients and patients with skin diseases or coagulopathy. Using the TS-SeV vector of the present invention, iPS cells can be produced with high efficiency of “˜4%” for dermal fibroblasts and “˜2%” for peripheral blood cells.
Operations such as infection of cells with the TS-SeV vector of the present invention, culture, treatment, and selection of infected and reprogrammed cells can be performed according to standard methods.
In addition, when cells are initialized using the TS-SeV vector of the present invention, the introduced gene or a part thereof is not inserted into an unspecified site of the chromosome. Furthermore, one embodiment of the TS-SeV vector of the present invention does not use a c-Myc gene or L-Myc gene having tumorigenic activity. For this reason, the obtained iPS cells are extremely safe without causing canceration.

  The vector carrying the reprogramming gene can be removed by shifting (increasing) the cell culture temperature. For example, cells that have been cultured and passaged at 37 ° C. are brought to a temperature above 37 ° C., preferably above 37 ° C. and below 39 ° C., more preferably 38 ° C. ± 0.5 ° C., and even more preferably 38 ° C. By increasing the temperature, the vector can be completely removed. The culture period at a temperature exceeding 37 ° C. is not particularly limited as long as the vector can be removed. However, for example, 2 to 20 days, preferably 2 to 15 days, more preferably 3 to 5 days, On the other hand, in terms of the passage number, it is preferably 1 to 3 passages, more preferably 1 to 2 passages. The treated cells are treated at a temperature exceeding 37 ° C., and then a single clone is separated to obtain a clone from which the vector is completely removed. Confirmation of vector removal can be performed by detecting any gene in the vector by a conventional method, for example, by RT-PCR. Thus, by using the TS-SeV vector of the present invention, iPS cells containing no transgene can be produced in a short period of time within one week after isolation of iPS cell colonies. Also, unlike conventional techniques that do not use SeV vectors, this system does not require multiple infection cycles, and iPS cell production efficiencies are similar to those of retroviruses, lentiviruses, or plasmid vectors, for example. 20 to 100 times that obtained using a simple technique.

IPS cells prepared from cells of patients with intractable diseases using TS-SeV of the present invention can exhibit disease characteristics (phenotype). The phenotype can be confirmed by inducing differentiation of the produced iPS cells into a desired cell lineage. Induction of differentiation of iPS cells can be performed according to a standard method. For example, differentiation induction into the liver lineage can be performed by culturing the prepared iPS cells in a hepatocyte differentiation induction medium. Thus differentiated cells show the characteristics (phenotype) of the disease from which the cells are derived. For example, hepatocyte-like cells derived from iPS cells from Niemann-Pick disease type C patients accumulate cholesterol and consequently exhibit dysfunction. Examples of the dysfunction include dysfunction of autophagy and ATP production. As another example, liver-like cells derived from iPS cells derived from a patient with GM-1 gangliosidosis exhibit a characteristic (phenotype) of autophagy abnormality.
Since the cells induced to differentiate from iPS cells derived from patients with refractory diseases become a cell model of the disease, they are a powerful tool for research and drug candidate screening.
For example, as described in detail in the Examples below, both 2-hydroxy-γ-cyclodextrin (HPGCD) and 2-hydroxypropyl-β-cyclodextrin (HPBCD) accumulate in hepatocyte-like cells derived from NPC. Cholesterol was removed to restore the function of hepatocyte-like cells. This indicates that HPGCD is a promising new candidate for the treatment of NPC.

The pharmaceutical composition of the present invention contains hydroxypropyl-γ-cyclodextrin as an active ingredient and can be used as a therapeutic agent for lysosomal disease. Examples of lysosomal diseases include Niemann-Pick disease, Tay-sachs disease, sialidosis, or GM-1 gangliosidosis.
As shown below, HPGCD showed an activity equal to or higher than that of HPBCD against hepatocyte-like cells induced to differentiate from NPS patient-derived iPS cells, which showed a Niemann-Pick disease phenotype. On the other hand, 2-hydroxy-α-cyclodextrin (HPACD) showed no activity. This result is based on a report confirming the cholesterol-lytic activity of HPBCD and HPGCD using cultured cells (Non-Patent Document 5), considering that the effect of HPGCD is less than one tenth of that of HPBCD. It ’s amazing.
One of the most important requirements for a drug candidate is the absence of endogenous cytotoxicity and an acceptable low level. The interaction between cyclodextrin and the cell membrane is an early stage of such cell damage. The in vitro lytic activity of isolated erythrocytes is an indicator of the toxicity of each cyclodextrin, but the hemolytic activity of hydroxypropylcyclodextrin is in the order of HPBCD>HPACD> HPGCD (Non-patent Document 5). Previous studies have shown that γ-cyclodextrin is safer than α- or β-cyclodextrin in acute intravenous administration to rats, lethal to 50% of the population (LD50 value) Are reported to be 1000, 788, and> 3750 mg / kg for α-, β-, and γ-cyclodextrin, respectively. Therefore, HPGCD is superior to HPBCD as a therapeutic agent for Niemann-Pick disease.

  The composition of the present invention is not limited to this, but preferably takes the form of an injectable preparation. The injectable preparation of the present invention can be administered intravenously, intramuscularly or subcutaneously. In addition, the pharmaceutical composition of the present invention can take any form of a water-soluble preparation or a lyophilized preparation, preferably an aqueous injection or a freeze-dried injection upon use.

  The composition of the present invention may contain saccharides, preservatives, stabilizers and antistatic agents which are usually used for injections. The composition of the present invention may also contain a pharmacologically acceptable pH adjuster. The pH adjuster used in the present invention is not particularly limited as long as it is a pharmacologically acceptable substance that can be used for pharmaceutical purposes, but preferably sodium hydroxide, carbonate buffer, phosphate buffer, Citrate buffer, acetate buffer and hydrochloric acid. These pH adjusters may be used alone or in combination of two or more. The composition of the present invention can also contain an osmotic pressure adjusting agent or an isotonic agent, and can contain, for example, at least one of sodium chloride, dextrose and the like.

The effective dose of the pharmaceutical composition of the present invention can be appropriately selected depending on the type of disease, the degree of illness, the treatment policy, the body weight, age, sex, and the (genetic) racial background of the patient. The effective amount is generally determined based on factors such as clinically observed symptoms and the degree of disease progression. The daily dose is, for example, 0.1 g / kg to 10 g / kg (3 g to 600 g for an adult with a body weight of 60 kg), preferably 0.2 g / kg to 10 g / kg, more preferably 0.2 g / kg. kg to 5 g / kg, more preferably 0.2 g / k to 2 g / kg. Administration may be carried out in a single dose or divided into multiple doses, and may be administered continuously over time by infusion or the like, preferably several hours or more by infusion, for example, Administration may take several hours to about 10 hours. The administration may be daily or intermittent, and can be appropriately selected depending on the condition of the administration subject, but is preferably intermittent administration. For example, 0.5 g / kg to 10 g / kg can be administered 1 to 3 times a week.
Moreover, since the pharmaceutical composition of the present invention is excellent in safety, it can be administered for a long time. That is, the lysosomal disease targeted by the pharmaceutical composition of the present invention is a genetic disease, and administration is often required as long as the patient survives. Since the pharmaceutical composition of the present invention is excellent in safety, it is particularly excellent in such use. The period during which the pharmaceutical product of the present invention can be administered is not particularly limited. However, the pharmaceutical product of the present invention can be administered over a long period of time, such as at least several weeks or more, preferably several months or more, more preferably multiple years or more. Can be administered.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to a following example.
1. Materials and Methods (1) Preparation of Sendai virus (SeV) vector Preparation and production of a temperature-sensitive Sendai virus vector were performed according to a report by Ban et al. A conventional SeV vector having Oct3 / 4, Sox2, Klf4, and c-Myc was also prepared according to a report by Fuchi et al. (Non-patent Document 2). To create the TS12 vector, three mutations, including D433A, R434A and K437A, were introduced into the polymerase related gene (P). To create a TS15 vector, two other mutations, L1361C and L1558I, were inserted into the polymerase-related gene (L) of TS12. The “three-in-one” vector inserted the human KLF4, OCT3 / 4, and SOX2 genes in order between the coding regions of the P and M genes as described in FIG. 1A. Each gene was flanked by E (endo), I (intervening) and S (start) sequences.

(2) Maintenance of human iPS cells Human iPS cells consist of 20% KNOCKOUT ™ serum replacement (KSR, Invitrogen), 2 mM L-glutamine (Life Technologies), 0.1 mM non-essential amino acids (NEAA, SIGMA) , DMEM / F12 supplemented with 0.1 mM 2-mercaptoethanol (SIGMA), 0.5% penicillin and streptomycin (Nacalai Tesque, Japan) and 5 ng / ml basic fibroblast growth factor (bFGF, WAKO, Japan) Maintained on MMC-treated MEF feeder cells in human iPS medium containing (SIGMA).

(3) Differentiation into hepatocyte-like cells For induction of hepatocyte-like cells (HLC), iPS culture medium was extracted from semi-confluent human iPS cell medium with 2% B27 (life technology), 100 ng / ml activin. Switched to embryonic endoderm differentiation medium containing RPMI1640 supplemented with A and 1 mM sodium butyrate (NAB, Sigma). The NaB concentration was changed to 0.5 mM on the second day. Cells are harvested on day 4 and contain DMEM supplemented with 20% KSR, 1 mM glutamine, 1 mM NEAA, 0.1 mM 2-mercaptoethanol (SIGMA), 1% dimethyl sulfoxide (DMSO, SIGMA) The seeds were replated on a matrigel-coated dish in liver differentiation medium. CXCR4 expression was examined by FACS on day 4. Cells were collected on day 11, 8.3% FBS, 8.3% tryptose phosphate broth (SIGMA), 10 mM hydrocortisone 21-hemisuccinate (SIGMA), 1 mM insulin (SIGMA), 2 mM glutamine, 10 ng / ml Re-cultured in liver maturation medium containing L15 medium (SIGMA) supplemented with 20 mg / ml of oncostatin M (OSM, R & D) and hepatocyte growth factor (HGF, R & D). On day 18, the cells were used for various experiments. For hydroxypropylcyclodextrin treatment, HLCs were cultured with 0.1 mM or 1 mM hydroxypropylcyclodextrin for 4 days. For annexin and TUNEL staining, HLCs were cultured from day 18 for 4 days or 1 week, respectively.

(4) Karyotype analysis Chromosome G-band analysis was performed according to the manufacturer's procedure at the request of Nippon Gene Laboratory Co., Ltd. (Sendai).

(5) Teratoma formation
Healthy volunteer and patient-derived iPS cell lines grown on the MEF feeder layer were collected by collagenase IV treatment and injected into the testes of NOD-SCID immunodeficient mice. Palpable tumors were observed about 8-12 weeks after injection. Tumor samples were collected, fixed with 10% formalin, embedded in paraffin, and hematoxylin-eosin stained according to standard procedures.

(6) RNA isolation and PCR
Total RNA was purified using Sepazol® Super G reagent (Nacalai Tesque, Japan). Total RNA was transcribed into DNA using Superscript III (Invitrogen) and random primers (Invitrogen). RT-PCR was performed using QuickTaq ™ (TOYOBO, Japan) according to the method described in Hamasaki et al. (Stem Cells, 30, 2437-2449, 2012). The primers used for Oct3 / 4, Sox2, Klf4 and c-Myc were designed to detect endogenous gene expression but not the transgene. To detect the Sendai virus genome, nested RT-PCR was performed. Primer sequences and amplification conditions are shown in Table 1 below (referred to as SEQ ID NO: 1 to SEQ ID NO: 48 from the top).

(7) Genome sequencing
The mutation of the NPC1 gene in the NPC-derived iPS cell line was confirmed by direct sequencing. The extracted genomic DNA was amplified by PCR, and the resulting PCR product was sequenced by ABI PRISM ™ 310 Genetic Analyzer (Applied Biosystems). Sequencing primers and amplification conditions are shown in Table 2 below (referred to as SEQ ID NO: 49 to SEQ ID NO: 56 from the top).

(8) Cell staining and immunocytochemistry Alkaline phosphatase staining was performed using leukocyte alkaline phosphatase kit (SIGMA). For immunocytochemistry, cells were fixed in PBS containing 4% paraformaldehyde for 30 minutes at 4 ° C. Samples were treated with 0.2% Triton X-100 for 15 minutes at room temperature (RT) for detection of molecules localized in the nucleus. Cells were washed 3 times with PBS containing 2% FBS and then incubated overnight at 4 ° C. in PBS containing 2% FBS with primary antibody. Nuclei were stained with propidium iodide (PI, WAKO, Japan) and 1 mg / ml Hoechst 33258 (Invitrogen). A list of primary and secondary antibodies is listed in Table 3 below. For Filipino staining, samples were washed three times with PBS after fixation and incubated for 10 minutes at room temperature in PBS containing 1.5 mg / ml glycine. Samples were then treated with PBS containing 10% FBS and 50 mg / ml Philippines (SIGMA). Data were calculated by UV absorption (360/460) and analyzed using Developer Toolbox software from IN CELL ANALYZER 6000 (GE Healthcare). The number of insoluble P62 granules was counted with IN CELL ANALYZER 6000 (GE Healthcare). To examine glycogen accumulation, periodate-Schiff (PAS) staining of hepatocyte-like cells was performed using PAS staining solution (Mudo Chemicals, Tokyo, Japan) according to the manufacturer's protocol.

(9) Immunoblot analysis Protein lysates were separated by SDS-PAGE and transferred to a PVDF membrane. LC3-I and LC3-II were detected by anti-LC3 antibody (cell signaling). Data were normalized by α-tubulin expression. After HLCs were dissolved in RIPA buffer, insoluble P62 was collected as a pellet after centrifugation of the sample.

(10) Albumin production analysis Albumin production of hepatocyte-like cells was measured with a human albumin ELISA quantification kit (Betil E80-129) according to the manufacturer's protocol. Data was normalized to the percentage of albumin positive in the sample.

(11) Cell size analysis The cell size of albumin positive cells was calculated by the developer toolbox software of IN CELL ANALYZER 6000 (GE Healthcare).

(12) Indocyanine Green (ICG) Analysis Cultured cells on differentiation day 18 were treated with 1 mg / ml ICG for 30 minutes at 37 ° C. Cells were washed 3 times with PBS and positive cells were analyzed. The cells were then incubated in medium for 5 minutes and analyzed again.

(13) ATP measurement
Hepatocyte-like cells derived from the iPS cell line were cultured in DMEM medium in the absence of glucose for 24 hours and then in DMEM medium containing 10% FBS and high glucose for 6 hours. ATP was measured with an ATP measurement kit (TOYO INK) according to the manufacturer's protocol.

(14) Mitochondrial staining with MitoTrackers
Hepatocyte-like cells derived from the iPS cell line were cultured for 20 minutes in the presence of 100 nM MitoTracker red CMXRos (Molecular Probes) and analyzed by FACS. HLCs were stained with JC-1 according to the manufacturer's protocol (Molecular Probe). The red and green fluorescence intensities of JC-1 staining were measured with the developer toolbox software of IN CELL ANALYZER 6000 (GE Healthcare).

(15) TUNEL staining
TUNEL staining was performed with the APO-BrdU TUNEL assay kit (Invitrogen) according to the manufacturer's protocol.

(16) Ammonia removal and urea secretion activity
HLCs were cultured for 2 days in medium containing 1 mM ammonium chloride. The supernatant was collected and the ammonia and urea concentrations were then measured by ammonia assay kit (SIGMA) and urea colorimetric assay kit (Biovision), respectively, according to the manufacturer's protocol.

(17) Cyclodextrin 2-hydroxypropyl-α-cyclodextrin (HPACD) having an average degree of substitution of 5.0, 2-hydroxypropyl-β-cyclodextrin (HPBCD) having an average degree of substitution of 4.7, and 2-hydroxy having an average degree of substitution of 6.4 Propyl-γ-cyclodextrin (HPGCD) was obtained from Nippon Shokuhin Kako (Tokyo, Japan).

(18) Antibody staining and FACS analysis Differentiated iPS cells were collected on day 4 and stained with biotin-conjugated mouse anti-human CXCR4 antibody (R & D Systems) and streptavidin-allophycocyanin (SA-APC, eBioscience). The percentage of apoptotic cells and dead cells was measured with a flow cytometer using annexin (Beckman Coulter) and 7-amino-actinomycin D (7-AAD, Beckman Coulter).

2. result
Example 1: Preparation of vector iPS cells were prepared using temperature-sensitive Sendai virus (SeV) vectors each having the sequence of four reprogramming factors (OCT3 / 4, SOX2, KLF4 and c-MYC).
In order to improve the production efficiency of iPS cells and reduce the time that the vector remains in the cells, the present inventors have developed three of the above factors, KLF4 (K), OCT3 (O) and SOX2 ( A new Ts-SeV vector, TS12KOS, with a coding sequence in which S) is connected in tandem in the KOS direction was created (Fig. 1). The TS12KOS vector contains three mutations that generate alanine residues (D433A, R434A, and K437A) within the large protein (L) binding domain of phosphorylated proteins, a component of Sendai virus RNA polymerase. SeV with these three mutations shows moderate expression of GFP at 37 ° C, but weak expression at temperatures above 38 ° C.

Example 2: Generation of iPS cells using TS-SeV vector Fibroblasts from healthy volunteers and patients were obtained after informed consent under a protocol approved by the ethics committee of each institution of the inventor. Isolated and prepared from a tissue piece of skin biopsy. Skin samples were minced and cultured in Dulbecco's modified essential medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS). After fibroblasts appeared, they were used for iPS cell induction.
Mononuclear cells (MNCs) were isolated by Ficall gradient for iPS cell formation from peripheral blood cells. To stimulate T lymphocytes, MNCs were cultured on dishes coated with anti-CD3 antibody with IL-2 for 5 days.
iPS cells were generated from human skin-derived fibroblasts and stimulated T lymphocytes according to the report of Seki et al. (2010). Briefly, 1 × 10 ⁵ human MNCs per well of a 48-well plate and 5 × 10 ⁵ human fibroblasts per well of a 6-well plate were seeded one day before infection and then 3, 10 Sendai virus (SeV) vectors were infected at multiple infectivity (MOI), including 30 and 30. Blood cells were cultured for 2 days, fibroblasts were cultured for 7 days, infected cells were harvested with trypsin, and 5 × per 60 mm dish on mouse embryo fibroblast (MEF) feeder cells treated with mitomycin C (MMC). 10 Reseeded with ⁴ cells. The next day, the medium was replaced with human iPS cell medium. New Sendai virus-infected cultures were cultured for 1 week at 36 ° C. 18-25 days after infection, colonies were picked and cultured again in human iPS cell medium. In order to remove Sendai virus, the culture temperature was changed from 37 ° C to 38 ° C in 1 or 2 passages of iPS cells.

  First, the production efficiency of iPS cells from human skin fibroblasts of healthy volunteers was compared between TS12KOS and a conventional SeV vector (FIG. 2). On the 28th day after induction, the number of colonies that showed alkaline phosphatase (AP) positive staining and a human embryonic stem (ES) -like morphology was counted. The production efficiency of iPS cells was significantly higher when the TS12KOS vector was used compared to the conventional vector.

  Next, the effect of temperature change on iPS cell production from human fibroblasts was examined. When the culture temperature was changed from 37 ° C to 36 ° for the first 2 weeks after infection, the colony production efficiency was maintained at a high level, but when the temperature was lowered for 3 weeks or more after infection, Efficiency was significantly reduced (Figure 3). Also, temperature changes were more effective during the first and second weeks after infection than in later periods. Therefore, the temperature drop for the first week only was used for the following experiments.

Example 3: Analysis of prepared iPS cells
Nested RT-PCR analysis of viral RNA was performed to determine whether the TS12KOS vector was removed from iPS cells more easily than conventional SeV vectors. Individual colonies were expanded and the temperature was shifted from 37 ° C to 38 ° C for 3 days at various passages. In conventional SeV infection, increasing temperature at passage 1 or 2 did not induce virus removal. In contrast, in the case of the TS12KOS vector, the temperature increase at passage 1 or 2 was 84% and 65% of virus genome negative iPS cell-like clones, respectively (FIG. 4). These results indicate that the TS12KOS vector is superior to the conventional SeV vector in terms of both iPS cell formation efficiency and virus removal from iPS cells.

Example 4: Production of iPS cells from human peripheral blood cells One goal is the development of safe and efficient vectors for forming iPS cells from human peripheral blood cells. Peripheral T lymphocytes were stimulated with both anti-CD3 antibody and interleukin 2, and then various SeV vectors were infected to prepare iPS cells. iPS cell production was significantly more efficient when using the TS12KOS vector than the conventional SeV vector (FIG. 5A). In conventional SeV infection, any change in temperature from 37 ° C. to 38 ° C. at passage 1 or 2 did not induce elimination of the vector from the iPS cell line. In contrast, when the TS12KOS vector was used under the same conditions, 65% and 47% of the clones were negative for the viral genome, respectively (FIG. 5B). Thus, similar to the results obtained with fibroblasts, removal of the TS12KOS vector from peripheral T lymphocyte-derived iPS-like cells was also faster than observed for conventional SeV vectors.

  Colonies formed from dermal fibroblasts and peripheral blood cells induced by the TS12KOS vector showed typical ES cell-like morphology and expressed a set of typical markers of pluripotency (data not shown). These iPS cell lines had normal 46 XY karyotypes even after increasing temperature and culturing for more than 10 passages (data not shown). To confirm the pluripotency of the clonal strain, one cell line was transplanted into the testis of immunodeficient mice. After 12 weeks of transplantation, the tested iPS cell lines formed teratomas, which contained all types of inducers of the three germ layers (FIG. 6). That is, the iPS cell line formed with the TS12KOS vector satisfied the criteria for iPS cells.

Example 5: Generation of iPS cell line showing disease phenotype To confirm the use of disease-derived iPS cells as a cell model, Niemann-Pick disease C, a lysosomal storage disease associated with mutations in NPC1 and NPC2 genes Targeted type (NPC). NPC1 functions as a transporter between endosomes and lysosomes, and NPC2, in cooperation with NPC1, transports intracellular molecules. Mutations in the NPC1 and NPC2 genes disrupt this transport system, leading to the accumulation of free cholesterol and glycolipids in lysosomes. An iPS cell line was generated from skin fibroblasts of two patients with different NPC1 mutations using the TS12KOS vector. The production efficiency of iPS cells from these patients was similar to that from healthy volunteers. The NPC-derived iPS cell line showed an ES cell-like morphology (FIG. 7) and expressed a set of pluripotency markers (FIG. 8). From the nested RT-PCR analysis, the iPS cell line was negative for SeV (FIG. 8).
The differentiation potential of the NPC-derived iPS cell line was then examined by evaluating teratoma formation. As a result of histological analysis, the teratomas analyzed consisted of the progeny of all three germ cell layers (all three germ layers) such as cubic epithelium, melanin pigment, cartilage, muscle, and various glandular structures ( FIG. 9). The prepared iPS cell line had normal karyotypes 46XY and 46XX (data not shown). Mutations in the NPC1 gene were confirmed by DNA sequencing (Figure 10). Thus, the NPC-derived iPS cell line met the criteria for iPS cells.

Example 6: Analysis of NPC-derived iPS cell line Hypertrophy of the liver is one of the main symptoms of NPC patients, and severe ones of the disease result in liver dysfunction and liver failure. To examine the effect of Npc1 deficiency on the hepatocyte lineage, the NPC-derived iPS cell line was differentiated into hepatocyte-like cells expressing albumin. From previous studies, activin A treatment selectively differentiates mouse ES cells into embryonic endoderm cells and hepatocyte-like cells (HLCs), and the endoderm surface marker Cxcr4 has been shown to differentiate endoderm. Based on these, the culture conditions were modified and HLCs were easily generated from human iPS cells (FIG. 11). On differentiation day 18, HLCs express α-fetoprotein (˜65% of total cells), albumin (˜˜80% of total cells), and other liver markers (data not shown) and indocyanine Green (ICG) was absorbed and glycogen was stored (data not shown). The production rate of embryonic endoderm-like cells calculated as a percentage of Cxcr4-positive cells, and the liver differentiation efficiency calculated from the percentage of albumin-positive cells and the expression of the markers are between normal iPS cells and NPC-derived iPS cell lines. It was the same. In contrast, the cell size of NPC-derived HLCs was larger than that of control HLCs (FIG. 12). In NPC patients, impaired lipid transport from endosomes to lysosomes results in the accumulation of free cholesterol in lysosomes. Therefore, Filipino staining was performed to detect free cholesterol in these cells, and the level of cholesterol accumulation was evaluated. In contrast, control HLCSs from healthy volunteers had very few positive staining cells, whereas in contrast, NPC-derived HLCs detected abnormal levels of cholesterol accumulation (FIG. 13). This indicates that these cells mirror the NPC cell phenotype.

  Next, various functions of HLCs derived from normal iPS cell lines and NPC-iPS cell lines were examined. No differences could be detected in terms of hepatocyte function, ICG uptake and release, glycogen storage, albumin production, urea secretion, or ammonia removal (data not shown). ATP levels in NPC-HLCs were significantly lower compared to control HLCs (FIG. 14). Nevertheless, apoptosis of NPC-HLCs was not worse compared to that of controls (data not shown). Subsequently, specific MitoTracker staining reagents JC-1 and CMXRos were used to examine the membrane potential of mitochondria. JC-1 is concentrated in mitochondria and aggregates at normal mitochondrial membrane potential, resulting in a high red / green fluorescence intensity ratio. Decreased mitochondrial membrane potential affects JC-1 aggregation and decreases the high red / green fluorescence intensity ratio. CMXRos accumulates in mitochondria at normal membrane potential. No difference in the staining pattern of JC-1 or CMXRos could be detected between normal and NPC-derived HLCs (data not shown).

  Cellular autophagy is impaired in lysosomal storage diseases. Two methods were used to monitor the autophagy pathway in control and NPC-derived HLCs. First, the expression of microtubule-associated protein 1 light chain 3 (LC3), which is a marker protein for autophagy, was examined. LC3 C-terminal treatment yields LC-I, which is modified to LC-II upon initiation of autophagosome formation. Subsequently, the expression of p62 / SQSTM1 (P62) was measured in order to evaluate autophagic flux. Since P62 binds to LC3 and is degraded when fused to lysosomes, autophagy flux dysfunction causes accumulation and aggregation of insoluble P62. The expression levels of LC3-II and insoluble P62 protein were higher in NPC-HLCs than in normal HLCs (FIGS. 15 and 16). Excessive P62 aggregation was also significantly observed in NPC-derived HLCs compared to normal HLCs (FIG. 17). These results suggest that autophagy was up-regulated with NPC-derived HLCs and that autophagy flux was impaired with NPC-derived HLCs.

Example 7: Effect of various cyclodextrin treatments on cholesterol accumulation and cell function recovery As mentioned above, NPC-derived iPS cells show NPC phenotype, so screening drug candidates for treatment of NPC An in vitro system is provided. The extreme cholesterol accumulation observed in NPC iPS cell-derived HLCs allows us to investigate the effects of various drug treatments on this process.
Since 2-hydroxypropyl-β-cyclodextrin (HPBCD) has been reported to be effective in reducing the accumulation of cholesterol in NPC1-deficient cells, a series of 2-hydroxypropyl-cyclodextrins with different cavity sizes can be used. The HLCs derived from normal and NPC-iPS cell lines were used to confirm the effect. HLCs were cultured for 4 days with 1 mM of each hydroxypropyl-cyclodextrin, then stained in the Philippines and then analyzed using IN CELL ANALYZER (GE Healthcare). In experiments using NPC-HLCs of the present invention, the observed cholesterol accumulation was significantly reduced with HPBCD treatment (FIG. 18). Interestingly, 2-hydroxypropyl-α-cyclodextrin (HPACD) had no effect on cholesterol accumulation, whereas 2-hydroxypropyl-γ-cyclodextrin (HPGCD) was observed with HPBCD. Decreased cholesterol accumulation.
The size of HLCs was reduced by treatment with HPBCD and HPGCD (data not shown). The effect of various concentrations of HPBCD or HPGCD on NPC-derived HLCs was confirmed. Cultivation with HPBCD or HPGCD for 4 days, followed by Philippine staining and then analysis with INCELL ANALYZER. Low concentrations (100 μM) of HPBCD and HPGCD had no effect on reducing cholesterol accumulation (FIG. 19). Next, treatment of cells differentiating into HLC with HPBCD was effective at an intermediate stage in liver differentiation (FIG. 20).

  Since NPC-derived HLCs showed abnormally low ATP levels and abnormal autophagy, it was next investigated whether cyclodextrin treatment could recover these abnormalities. HLCs were cultured with 1 mM hydroxypropyl-cyclodextrin for 4 days to confirm ATP levels, LC3 expression levels, p62 expression levels and insoluble p62 granules. As a result, treatment with HPBCD and HPGCD restored both ATP levels (FIG. 21) and autophagy function (FIGS. 22-24). As shown in FIG. 22, the expression level of LC3 was restored to a normal level by treatment with HPBCD and HPGCD, indicating that the abnormal induction of autophagy was restored. As shown in FIG. 23, treatment with HPBCD and HPGCD reduced the amount of insoluble p62. Moreover, as shown in FIG. 24, the ratio of HLCs having insoluble p62 aggregates exceeding 40 granules was greatly reduced by the treatment with HPBCD and HPGCD. These indicate that the failure of autophagy flux has been recovered. This indicates that HLCs derived from NPC-iPS cell lines are useful for evaluating drug candidates. In addition to HPBCD, HPGCD has also been shown to be a drug candidate for the treatment of NPC.

Example 8: Effect of HPBCD and HPGCD on NPC-derived HLCs
In order to confirm that the mechanism of action of HPBCD and HPGCD on NPC-derived HLCs (recovery of ATP levels and recovery of autophagy function) is the same, microarray analysis of HLCs treated with HPBCD and HPGCD (cluster analysis and Principal component analysis (PCA) was performed. Liver-like cells (HLCs) induced by the method of FIG. 11 were cultured for 4 days with addition of HPBCD or HPGCD (control without addition of HPCD). The RNA was extracted and the gene expression was analyzed with a DNA microarray. Specifically, the procedure was as follows.
250 ng of total RNA from iPS-derived HLCs cultured under each condition was fragmented with biotin using 3′IVT Express kit (Affymetrix) according to the manufacturer's protocol. The sample was then hybridized to GeneChip® Human Genome U133 Plus 2.0 (Affymetrix) and the array was scanned with a GeneChip® Scanner 3000 (Affymetrix). Data was analyzed using GeneSpring GX 12.5 software (Agilent technologies). Each chip was normalized to the average of the measured values. Those in which the gene expression changes more than 1.5 times between NPC and normal cells were judged to be different. By comparing the profiles of genes with altered expression to each other, common up-regulated and down-regulated genes were identified in NPC-derived HLCs. Gene ontology biological processes were enhanced using gene set enrichment analysis (GSEA) (BROADINSTITUTE). This indicates a common up-regulated or down-regulated gene of NPC-derived HLCs, as well as different gene expression in HPBCD and HPGCD treatment. An algorithm was used that was performed 1000 times for statistical analysis and enhanced the biological process of gene ontology including genes that appeared more than 5 times. Gene ontology biological processes were selected and described only based on p-value ranking. A p value of <0.05 or <0.1 means that NPC or HPGCD treatment is significantly changed relative to normal or HPBCD treatment, respectively. Hierarchical cluster analysis was then performed to identify genes that were prominent in biological processes.

  Clusters of normal (N1), NPC-5 (A114), and NPC-6 (A225) fibroblasts (fibro), iPS cells, and HLCs derived from them in the absence of HPCD, HPBCD or HPGCD The results of the analysis are shown in FIG. 25, and the results of the PCA analysis are shown in FIG. This shows that HPGCD works by a mechanism different from HPBCD.

  FIG. 27 shows signature molecules changed in NPC-derived HLCs, which were confirmed as a result of the above analysis. The upper panel shows molecules that are significantly down-regulated in NPC, and the lower panel shows molecules that are significantly up-regulated in NPC.

  The effect of HPBCD or HPGCD treatment on the expression of the signature molecule was confirmed. The results are shown in FIG. The upper panel shows the effect of HPBCD processing, and the lower panel shows the effect of HPGCD processing.

  Hierarchical cluster analysis confirmed the genes that markedly changed by HPCBD or HPGCD treatment. The results are shown in FIG. In FIG. 28, the signature molecule gene data set enclosed in a square was clustered according to Euclidean distance metrics. The expression pattern of signature molecules altered by HPGCD treatment was closer to that of normal human HLCs than by HPBCD treatment.

Example 9: Effect of HPGCD treatment on NPC model mice
The effect of HBGCD on cholesterol accumulation in NPC-derived HLCs was confirmed using NPC model mice. The model mouse (NPC mouse) is deficient in the transport of cholesterol from lysosome to ER due to spontaneous mutation of Npc1 gene. This model mouse also exhibits symptoms similar to those of human diseases with cholesterol accumulation in the liver and brain. This model mouse exhibits liver damage and nerve dysfunction and dies at 12 weeks of age without treatment.
Four-week-old NPC mice were treated with HPCGD (4000 mg / kg) every week starting from the age of four weeks, and samples were collected at 8.5 weeks (all treated five times). The control was treated with saline. The experiment was repeated twice (first time: n = 6, second time: n = 4).

When serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase), which are markers of liver damage, were measured, they were markedly decreased by HPGCD treatment. The results are shown in FIG. As a result of histological analysis, the morphology of the liver of NPC mice was remarkably improved by the HPGCD treatment (FIG. 31). In addition, the HPGCD treatment also recovered the deletion of Purkinje cells in the middle cerebellum of NPC mice (FIG. 32).
In addition, HPGCD-treated NPC mice recovered abnormal autophagy, and in addition, the expression levels of LC3 and insoluble p62 were restored to normal levels in the liver and brain of HPGCD-treated NPC mice (data not shown). ).
In order to confirm the effect of HPGCD treatment on the survival rate of NPC mice, HPGCD was administered to model mice every week from 4 weeks of age (HPGCD administration group: n = 6, control (saline administration) Group): n = 6). When the survival curve was confirmed, a remarkable improvement in the survival rate was observed. The results are shown in FIG.

Example 10: Toxicity test of HPBCG and HPGCD
To confirm the superior safety of HPGCD, acute toxicity to normal mice was tested. 14.4 mM HPBCD and HPGCD were each administered at 19.18 ml / g into the subcutaneous tissue of 8-week-old mice (n = 10), and the survival rate was confirmed. Mice that received HPBCD almost died 72 hours after administration, but no death was confirmed in mice that received HPGCD.

The above results described herein indicate that iPS cell lines without transgenes are effective cell models for intractable diseases.
We have also generated more than 1000 iPS cell lines from over 100 patients with refractory disease so far using SeV vectors containing TS12KOS vectors. An example is shown in Table 4. In the table, Miyoshi Myopasy uses the previous vector instead of the improved TS12KO vector of the present invention. All iPS cell lines generated from these patients showed ES cell-like colony morphology and expressed a set of pluripotency markers (data not shown). Moreover, the SeV negative state of all established iPS cell lines was confirmed by nested RT-PCR (data not shown). This indicates that these generated cell lines do not carry the transgene used for reprogramming.

  The above description is merely illustrative of the objects and objects of the present invention and is not intended to limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein without departing from the scope of the appended claims.

The pharmaceutical composition containing the hydroxypropyl-γ-cyclodextrin of the present invention as an active ingredient is useful as a therapeutic agent for lysosomal disease, particularly Niemann-Pick disease or GM-1 gangliosidosis.
Moreover, the iPS cell and the screening method of the present invention can be used for screening therapeutic drugs for intractable diseases.

Claims (4)

  A pharmaceutical composition for treating or preventing lysosomal disease, comprising hydroxypropyl-γ-cyclodextrin as an active ingredient.   The pharmaceutical composition according to claim 1, wherein the lysosomal disease is Niemann-Pick disease.   The pharmaceutical composition according to claim 1, wherein the lysosomal disease is GM1 gangliosidosis. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is an injection and is administered for a period of several weeks or more .